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Lycium barbarum geranyl geranyl pyrophosphate synthase gene and its encoded protein and application

A technology of gene coding and pyrophosphate, which is used in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2016-06-22
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] With the continuous discovery of the medicinal value and health care function of lycopene and carotenoids, the demand for the types and production of lycopene and carotenoids will also increase. However, lycopene carotenoids Difficult to synthesize chemically

Method used

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  • Lycium barbarum geranyl geranyl pyrophosphate synthase gene and its encoded protein and application
  • Lycium barbarum geranyl geranyl pyrophosphate synthase gene and its encoded protein and application
  • Lycium barbarum geranyl geranyl pyrophosphate synthase gene and its encoded protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Cloning of the geranylgeranyl pyrophosphate synthase gene LmGGPS2 from Lycium barbarum

[0047] Total RNA was extracted from fresh leaves of Lycium barbarum, and the RNA was reverse-transcribed into cDNA using the 3'FULLRACECoreSetVer.2.0 (TaKaRa, Japan) kit. The specific steps refer to the instructions. The reaction system is as follows:

[0048] RNA

[0049] Reaction conditions: 42°C, 60min; 70°C, 15min.

[0050] Design upstream primers LmGGPS-F1:5'-GTCATGTCTATGCTAATAGGTGT-3', LmGGPS-R1:5'-GGTCAGTTTCTGATGGAGAAATT-3', Using the synthesized cDNA as a template, the PCR reaction system is as follows:

[0051] 1st Strand cDNA

[0052] A total of 4 tubes of reaction solution were prepared, and the reaction conditions were as follows: 94°C, 4min; 94°C, 30Sec; 56°C, 30Sec; 72°C, 1min20Sec; 72°C, 10min; 30 cycles. After the reaction, the PCR reaction product was purified using Tiangen Company's ordinary DNA product purification kit to obtain a purified Lm...

Embodiment 2

[0054] Construction process of cloning vector pMD18-T-LmGGPS2

[0055] The LmGGPS2 gene shown in the sequence listing is connected to the pMD19-T carrier, and the reaction system is as follows:

[0056] LmGGPS2 full-length recovery product

4μL

pMD18-T vector

1μL

Solution I

5μL

[0057] The LmGGPS2PCR fragment is the recovered GGPS full-length product in Example 1.

[0058] Reaction conditions: 16°C, 30min. The ligation product was transformed into competent E-Coli.TOP10, spread on LB agar plate medium containing 40ml25mg / ml X-Gal, 16ml50mg / ml IPTG, 100mg / LAmp and cultured to form a single colony. Pick white colonies, colony PCR method to confirm the length of the insert in the T vector, such as figure 2 , as expected, the vector was sent to Huada Genomics Company for sequencing, and we obtained the 1074bp base sequence of the gene, which was blasted at NCBI and showed high homology with Solanaceae, indicating that the gene was cloned...

Embodiment 3

[0060] Construction process of Escherichia coli expression vector pET28a-LmGGPS2

[0061] The Escherichia coli containing the pMD19-T-LmGGPS2 plasmid obtained in the expanded culture experiment example 2 was extracted, and the plasmid was double-digested with BamHI and SalI, and the pET28a empty vector was double-digested with BamHI and SalI, purified and ligated , to obtain the Escherichia coli expression vector pET28a-LmGGPS2, and connect the digested products of the two.

[0062] The connection system is as follows:

[0063] pET28a empty vector fragment

[0064] The ligation product was transformed into competent Escherichia coli BL21. Spread on LB plates containing 100 mg / L kana resistance, and culture at 37°C. After 12 hours, pick a single colony for colony PCR verification, such as image 3 , Shake the colony PCR-positive bacteria, extract the plasmid, digest and identify the target band, and finally send it to Huada Gene Sequencing Company for sequencing. T...

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Abstract

The invention relates to a geranylgeranyl pyrophosphate synthetase gene in Lycium chinense Miller, and encoded protein and application thereof, in particular to a clone of the geranylgeranyl pyrophosphate synthetase gene LmGGPS2 in Lycium chinense Miller. Total RNA in fresh Lycium chinense Miller leaves is extracted, the geranylgeranyl pyrophosphate synthetase gene LmGGPS2 in Lycium chinense Miller is cloned, and the complete sequence of the obtained gene is 1246bp; an escherichia coli expression vector pET28a-LmGGPS2 is constructed, and LmGGPS2 expression protein is obtained through an escherichia coli heterologous expression system; a binary plant expression vector pCAMBIA2300-LmGGPS2 is constructed, the vector is transformed to agrobacterium C58 cells through an electric shock method, and tobaccos are transformed through the cells to obtained transgenic tobaccos. Tests find that the transgenic tobaccos greatly increase the content of ycopene of plant, and the content of carotene is also increased.

Description

technical field [0001] The invention relates to a wolfberry geranyl geranyl pyrophosphate synthase gene and its coded protein and application thereof, in particular to the cloning of the geranyl geranyl pyrophosphate synthase gene LmGGPS2 in Lycium chinense Miller. Background technique [0002] Geranylgeranyl pyrophosphate (GGPP) is an important intermediate metabolite ubiquitous in the biological world. In plants, GGPP participates in chlorophyll, carotenoids, gibberellins, plastoquinones, vitamin E, monoterpenes and benzoquinones, etc. The synthesis of products has an important impact on plant photosynthesis, growth and development, and product quality. The product was directly catalyzed by GGPS. GGPP is not only the most direct precursor of carotenoid biosynthesis, but also the precursor of gibberellin, chlorophyll and plastoquinone in plants. The synthesis of GGPP is the rate-limiting process in carotenoid synthesis. Through a series of enzymatic reactions, the lycope...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/70C12N1/21C12N5/10C12N15/82
Inventor 季静王罡贾翠翠吴电云曹海燕
Owner TIANJIN UNIV
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